The application of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA)-salt, to unilaterally nephrectomised rats induces severe hypertension due to volume-overload, and mimics human primary aldosteronism. Importantly, DOCA-salt hypertension is characterized by severe cardiac and renal lesions triggered by nuclear factor kappa B (NF-kappaB), activating protein (AP-1), and transforming growth factor beta1 (TGF-beta1) leading to end-stage organ damage. Although DOCA-salt hypertension is a low renin model, local production of angiotensin-II and aldosterone in cardiac and renal tissues stimulate TGF-beta1, fibronectin and collagen-1 causing fibrosis and hypertrophy. Since TGF-beta1 gene promoter contains binding sites for NF-kappaB and AP-1, cross-talk between TGF-beta1, NF-kappaBnand AP-1 can be envisaged. Accordingly, the activation of TGF-beta1, fibronectin, collagen, NF-kappaB and AP-1 may constitute a potent destructive force in hypertension.<p> Emerging evidence indicates that upregulation of the heme oxygenase (HO) system is cytoprotective with antioxidant, antihypertensive and antihypertrophic effects. Interestingly, the promoter region of HO-1 gene harbors consensus-binding sites for NF-kappaB and AP-1; therefore, the HO system may regulate these transcription factors to counteract tissue insults. However, the multifaceted interactions between the HO system, NF-kappaB, AP-1, TGF-beta1, fibronectin and collagen in mineralocorticoid-induced end-stage-organ damage have not been fully characterized. Similarly, the effect of the HO system on tissue angiotensin-II and aldosterone levels in mineralocorticoid-induced hypertension remains unclear. Therefore, the present study was designed to investigate the antihypertrophic effect of the HO system in cardiac and renal tissue of DOCA-salt hypertensive rats. In this study, the HO inducer, hemin, lowered blood pressure and attenuated cardiac/renal hypertrophy, whereas the HO inhibitor, chromium mesoporphyrin (CrMP), nullified the effects of hemin and exacerbated cardiac/renal injury the DOCA-salt hypertensive rats. The protective effect of hemin was associated with increased HO-1, HO activity, cyclic guanosine monophosphate (cGMP), superoxide dismutase activity, ferritin and the total antioxidant capacity in the cardiac and renal tissue. In contrast, angiotensin-II, aldosterone, 8-isoprostane, NF-kappaB and AP-1 were significantly downregulated. Furthermore, hemin therapy attenuated TGF-beta1 and extracellular matrix (ECM) proteins such as fibronectin and collagen, with corresponding reduction of cardiac histopathological lesions, including longitudinal/cross-sectional muscle fiber thickness, scarring, muscular hypertrophy, coronary arteriolar thickening and collagen deposition. Similarly, hemin attenuated structural lesions in the kidney such as glomerular hypertrophy, glomerular sclerosis, mononuclear cell infiltration, tubular cast formation, tubular dilation and renal arteriolar thickening with concomitant improvement of kidney function as evidenced by reduction of plasma creatinine, proteinuria, but enhanced creatinine clearance.<p> Collectively, these results suggest that the HO system suppressed hypertension, cardiac and renal fibrosis, and hypertrophy in the DOCA-salt hypertensive rat by downregulating transcription factors such as NF-kappaB and AP-1, reducing ECM proteins such as fibronectin and collagen, decreasing local tissue production of angiotensin-II and aldosterone, and improved renal functional capacity.

The application of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA)-salt, to unilaterally nephrectomised rats induces severe hypertension due to volume-overload, and mimics human primary aldosteronism. Importantly, DOCA-salt hypertension is characterized by severe cardiac and renal lesions triggered by nuclear factor kappa B (NF-kappaB), activating protein (AP-1), and transforming growth factor beta1 (TGF-beta1) leading to end-stage organ damage. Although DOCA-salt hypertension is a low renin model, local production of angiotensin-II and aldosterone in cardiac and renal tissues stimulate TGF-beta1, fibronectin and collagen-1 causing fibrosis and hypertrophy. Since TGF-beta1 gene promoter contains binding sites for NF-kappaB and AP-1, cross-talk between TGF-beta1, NF-kappaBnand AP-1 can be envisaged. Accordingly, the activation of TGF-beta1, fibronectin, collagen, NF-kappaB and AP-1 may constitute a potent destructive force in hypertension.<p> Emerging evidence indicates that upregulation of the heme oxygenase (HO) system is cytoprotective with antioxidant, antihypertensive and antihypertrophic effects. Interestingly, the promoter region of HO-1 gene harbors consensus-binding sites for NF-kappaB and AP-1; therefore, the HO system may regulate these transcription factors to counteract tissue insults. However, the multifaceted interactions between the HO system, NF-kappaB, AP-1, TGF-beta1, fibronectin and collagen in mineralocorticoid-induced end-stage-organ damage have not been fully characterized. Similarly, the effect of the HO system on tissue angiotensin-II and aldosterone levels in mineralocorticoid-induced hypertension remains unclear. Therefore, the present study was designed to investigate the antihypertrophic effect of the HO system in cardiac and renal tissue of DOCA-salt hypertensive rats. In this study, the HO inducer, hemin, lowered blood pressure and attenuated cardiac/renal hypertrophy, whereas the HO inhibitor, chromium mesoporphyrin (CrMP), nullified the effects of hemin and exacerbated cardiac/renal injury the DOCA-salt hypertensive rats. The protective effect of hemin was associated with increased HO-1, HO activity, cyclic guanosine monophosphate (cGMP), superoxide dismutase activity, ferritin and the total antioxidant capacity in the cardiac and renal tissue. In contrast, angiotensin-II, aldosterone, 8-isoprostane, NF-kappaB and AP-1 were significantly downregulated. Furthermore, hemin therapy attenuated TGF-beta1 and extracellular matrix (ECM) proteins such as fibronectin and collagen, with corresponding reduction of cardiac histopathological lesions, including longitudinal/cross-sectional muscle fiber thickness, scarring, muscular hypertrophy, coronary arteriolar thickening and collagen deposition. Similarly, hemin attenuated structural lesions in the kidney such as glomerular hypertrophy, glomerular sclerosis, mononuclear cell infiltration, tubular cast formation, tubular dilation and renal arteriolar thickening with concomitant improvement of kidney function as evidenced by reduction of plasma creatinine, proteinuria, but enhanced creatinine clearance.<p> Collectively, these results suggest that the HO system suppressed hypertension, cardiac and renal fibrosis, and hypertrophy in the DOCA-salt hypertensive rat by downregulating transcription factors such as NF-kappaB and AP-1, reducing ECM proteins such as fibronectin and collagen, decreasing local tissue production of angiotensin-II and aldosterone, and improved renal functional capacity.

Inibidores da Na+/K+ ATPase, como a ouabaína (OUA), estão associados à gênese/manutenção da hipertensão arterial (HA). Ratos tratados com OUA ficam hipertensos e suas artérias mesentéricas de resistência (AMR) apresentam disfunção vascular, estresse oxidativo e remodelamento estrutural e mecânico. Modelos de HA dependente de volume, como o DOCA-sal, têm aumento da concentração plasmática de OUA. Interagindo com a Na+/K+ ATPase, a OUA ativa vias de proliferação e fibrose, como a via MAPK, ativada quando da fosforilação da c-Src e do EGFR. A rostafuroxina, antagonista da OUA, reduz a pressão arterial (PA) e melhora a disfunção endotelial em AMR de ratos DOCA-sal. Dados do laboratório (não publicados) mostram que a rostafuroxina reverte o remodelamento mecânico e restaura parcialmente o remodelamento estrutural das AMR de ratos DOCA-sal. Assim, o objetivo deste trabalho foi avaliar os mecanismos de sinalização celular envolvidos nas mudanças estruturais e mecânicas das AMR de ratos DOCA-sal tratados com rostafuroxina. Ratos Wistar, uninefrectomizados, foram tratados com injeções subcutâneas de DOCA (em mg/kg/semana: 20 na primeira; 12 na segunda e terceira; 6 da quarta ao fim do experimento) e ingeriram água com NaCl (1%) e KCl (0,2%). Cinco semanas após o início do tratamento, um subgrupo passou a ser tratado, concomitantemente, com rostafuroxina (gavagem, 1mg/kg/dia) por 3 semanas. Ao final do protocolo, os animais foram anestesiados, mortos, as AMR dissecadas e fixadas em paraformaldeído (4%) para as análises histológicas ou armazenadas para Western Blot. Análise estatística aplicada: ANOVA uma ou duas vias. Avaliada por meio da plestismografia de cauda, o tratamento com DOCA-sal aumentou a PA; a rostafuroxina reduziu, mas não normalizou a mesma aos valores do grupo SHAM. A razão de colágeno I/III e a expressão proteica dos mediadores fibróticos TGF&beta;1 e CTGF foi maior nas AMR dos DOCA, alterações restauradas após tratamento com rostafuroxina. Além do mais, houve aumento da espessura da lâmina elástica interna das AMR dos DOCA, o que foi revertido pela rostafuroxina. A expressão de endotelina-1 foi maior nas AMR tanto dos DOCA tratados ou não com rostafuroxina; a expressão do receptor ET&Alpha; foi reduzida nas AMR de ambos os grupos. Já a expressão do receptor ET&Beta; foi maior nas AMR dos DOCA quando comparados aos SHAM, sendo normalizada pela rostafuroxina. A expressão de c-SrcTyr418 e EGFR, bem como das enzimas c-RAF, ERK 1/2, p-ERK 1/2 e p38MAPK foi maior nas AMR dos DOCA, sendo restauradas com a rostafuroxina. Os dados sugerem que o remodelamento das AMR do grupo DOCA pode ser atribuído à ação direta da OUA sobre a via fibrótica e sobre a elastina, interferindo na rigidez e no diâmetro interno, e também à sua ação indireta, mediada pela endotelina-1 que, agindo em receptores ETB e transativando o EGFR, ativa a via da MAPK, a qual está associada aos eventos de proliferação e fibrose celular. / Na+/K+ ATPase inhibitors, such as ouabain (OUA), are associated with genesis/maintenance of hypertension. Rats treated with OUA are hypertensive and their mesenteric resistance arteries (MRA) exhibit vascular dysfunction, oxidative stress and structural and mechanical remodeling. Volume-dependent hypertensive models, as DOCA-salt, have increased plasmatic OUA levels. Interacting with Na+/K+ ATPase, OUA activates proliferation and fibrosis pathways, such as MAPK pathway, which is activated from c-Src and EGFR phosphorylation. Rostafuroxin, an OUA antagonist, reduces arterial pressure (AP) and improves endothelial dysfunction in MRA of DOCA-salt rats. Unpublished data of our laboratory have shown that rostafuroxin reverses the mechanical and partially restores the structural remodeling of MRA of DOCA-salt rats. Thus, the aim of this study was to evaluate the cellular signaling mechanisms involved in the structural and mechanical changes in MRA of DOCA-salt rats treated with rostafuroxin. Male Wistar rats, uninephrectomized, were treated with subcutaneous injections of DOCA (mg/kg/week: 20 in the 1st; 12 in the 2nd and 3rd; 6 from 4th to the end of the experiment) and drunk water with NaCl (1%) and KCl (0,2%). After 5 weeks of treatment, a subgroup started being treated, concomitantly, with rostafuroxin (gavage, 1mg/kg/day) for 3 weeks. At the end of the protocol, animals were anesthetized, killed, the MRA dissected out and fixed in paraformaldehyde (4%) to histological analyses or stored to Western Blot. Statistical analysis applied: ANOVA one- or two-way. Evaluated by pletismography, the DOCA-salt treatment raised the AP levels; rostafuroxin reduced it, but did not normalized to the values of SHAM group. Collagen I/III ratio and protein expression of fibrosis mediators TGF&beta;1 and CTGF was higher in MRA of DOCA and rostafuroxin restored these parameters. In addition, an increment in the thickness of inner elastic lamina was observed in MRA of DOCA, which was reversed by rostafuroxin treatment. Endothelin-1 expression was higher in MRA of both DOCA and rostafuroxin treated rats; ET&Alpha; receptor was less expressed in both groups. On the other hand, ET&Beta; was more expressed in MRA from DOCA and normalized to SHAM levels by rostafuroxin treatment. The expression of c-SrcTyr418 and EGFR, as well as of the enzymes c-RAF, ERK 1/2, p-ERK 1/2 and p38MAPK was higher in MRA of DOCA when compared to SHAM, being restores with rostafuroxin. Taken together, the present data suggest that MRA remodeling in DOCA group can be attributed to direct action of OUA on elastin and fibrotic pathway, interfering in stiffness and inner diameter, as well as to its indirect action, mediated by endothelin-1, which acting in ETB receptors and activating EGFR, activates MAPK pathway, which is associated to proliferation and fibroses events.

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